A common goal of anti-cancer therapy and diagnostic imaging is the development of molecular constructs that can specifically target the tumor cells with minimal cross-reactivity to normal tissues. Cell surface receptors and epitopes are attractive targets for such agents as they are more accessible than are intracellular targets, allowing many more degrees of freedom in the design of the targeting agents. The current proposal seeks to develop targeting agents for Pancreatic Adenocarcinoma, PaAdo, a devastating disease with high mortality and no effective therapies. Data from our group and others suggest that a possible solution to this problem can come from the use of multimeric agents that are targeted to more than one receptor simultaneously. Multimeric ligands are a new class of targeting agents that contain multiple copies of binding moieties. Hence, they bind with cooperative affinity and binding can be non-linear with respect to target protein expression levels. In addition, they provide the possibility of mixing multiple different binding ligands onto the same chemical construct, providing a further level of discrimination. This project is organized around 3 independent specific aims that address fundamental vacancies in our knowledge: Appropriate targets on human cancers were inadequately identified. Over the past year, we have analyzed RNA expression data from 28 PaAdos and 103 normal tissues. From these data, we have identified 29 gene products that can potentially discriminate PaAdo from normal tissues in as single agents or in 3- and 4-gene combinations. Three of these targets have been validated by immunohistochemistry, IHC. The focus of Aim 1 will be to examine the remaining 26 gene products using IHC of tissue arrays. Small ligands are useful but not readily available. Although antibodies are useful targeting agents, assembling them into multimeric complexes can make them prohibitively large. Hence, we are identifying and iteratively developing smaller targeting ligands with soluble-phase and solid-support high-throughput methods. These methods will be applied in aim 2 for up to five of the validated targets. The structure-activity relationships (SAR) for multimeric constructs are ill-defined. Since this is a relatively new approach, the SAR for scaffolds and linkers is not well known. Work by us and others over the past few years has begun to develop a more solid understanding, but more data are needed before de novo engineering of such constructs will become viable. In Aim 3 we will investigate polymer scaffolds and linear linkers as divergent approaches to systematically define the degrees of freedom inherent in engineering such systems. The long-term goal of this work is to develop targeting agents appropriate for in vivo human use, which will carry payloads appropriate for diagnostic imaging and delivery of therapy.